Biomedical subjects

C Pueyo

Publications and source records attributed to C Pueyo.

At least 19 recordsLinked to original sources

PubMed2006

[Esophageal atresia type I. Is impossible possible?].

INTRODUCTION: Treatment of esophageal atresia with "long gap" remains difficult and controversial. According to the idea that esophageal anastomosis is imposible in most cases, several esophageal substitution methods have been proposed, as esophagocoloplasty, gastric transposition or reversed gastric tube. Nevertheless reconstruction of native esophagus is accepted as the best option if posible. "Long gap" definition is imprecise, expressed by variability in percent of these cases in total esophageal atresias reported in different series in literature. We report our experience in seven cases type I esophageal atresia with long gap and the different therapeutic options used, with attention to delayed or early esophageal anastomosis feasibility and outcome. MATERIAL AND METHODS: We have treated 121 patients with esophageal atresia from whom we analized 7 cases with pure esophageal atresia with "long gap" (5.8%). Six patients underwent gastrostomy and two gastrostomy and esophagostomy. Five patient underwent primary repair with esophageal anastomosis, delayed between 14 days and 4 months in 4 cases. One patient underwent esophageal anastomosis in the first day without gastrostomy. Retroesternal esophagocoloplasty was performed in 2 patients about their first year of life. Esophagogram was done in first month after surgery and pH monitoring of gastroesophageal reflux. Follow-up ranged from 6 months to 28 years. RESULTS: Esophageal anastomosis was feasible in all 5 patients in whom it was tried. Stricture occurred in two patients, one patient underwent anastomotic resection and new esophageal anastomosis. Esophageal reflux was present in two patients, one of them required funduplication. One patient was dead by complications of cardiac malformation. Remaining patients have normal swallowing and are in normal growth curves. Patients with esophagocoloplasty had not relevant early or late complications. CONCLUSIONS: In most pure esophageal atresia, delayed or even early esophageal anastomosis is feasible, making use of surgical and anesthesic sources that favour it. Esophageal substitution techniques can be reserved if this approach fails.

Anastomosis, Surgical↗

PubMed2003

Bone mass and bone modelling markers in hypertensive postmenopausal women.

Numerous phosphocalcium alterations associated with bone mineral density in hypertension have been described, but very few studies assess them. This study assesses bone mass in hypertensive postmenopausal women and the hypertension influence determining both calcium homeostasis and bone turnover markers. Blood and urine samples were analysed for calcium metabolism-related parameters. Densitometry studies were conducted in the lumbar spine (L2-L4). Hypertensive osteoporotic women--selected from 82 women, with 22% osteoporosis prevalence, similar to the rate for the same age in the Spanish population--had significantly higher levels of body mass index (29+/-4 vs 26+/-4, P=0.019), calciuria (293+/-146 vs 210+/-116 mg/24 h, P=0.023) and calcium/creatinine ratio (0.33+/-0.2 vs 0.22+/-0.1 P=0.003) vs hypertensive nonosteoporotic women. No relation was found between systolic and diastolic blood pressure with bone mass. However, there was a negative osteocalcin correlation (r=-0.386, P=0.0001, and r=-0.242, P=0.033). Calciuria is associated with bone mass decrease in hypertensive women, and there is no relation between bone mass and blood pressure.

Adult↗

PubMed2001

The effectiveness of the O(6)-alkylguanine-DNA alkyltransferase encoded by the ogt(ST) gene from S. typhimurium in protection against alkylating drugs, resistance to O(6)-benzylguanine and sensitisation to dibromoalkane genotoxicity.

Here we demonstrate that the Ogt(ST) from Salmonella typhimurium is a highly efficient O(6)-alkylguanine-DNA alkyltransferase (AGT) in affording protection against antitumour chloroethylating drugs (1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU)). In addition, Ogt(ST) is refractory to O(6)-benzylguanine (BG) inactivation and its expression provides only minor sensitisation to genotoxicity by environmental dibromoalkanes (DBE). No other of the assayed bacterial or human AGTs displayed such advantageous properties for chemoprotective gene therapy strategy. Our observations indicate that the Ogt(ST) AGT might be, under some circumstances, of potential use to improve cancer chemotherapy. At least, its properties may provide further insight into the design of human AGT variants that could be expressed in normal or tumour cells to provide either protection or ablation.

Alkanes↗

PubMed2001

Expression analysis of the nrdHIEF operon from Escherichia coli. Conditions that trigger the transcript level in vivo.

Escherichia coli has two aerobic ribonucleotide reductases encoded by the nrdAB and nrdHIEF operons. While NrdAB is active during aerobiosis, NrdEF is considered a cryptic enzyme with no obvious function. Here, we present evidence that nrdHIEF expression might be important under certain circumstances. Basal transcript levels were dramatically enhanced (25-75-fold), depending on the growth-phase and the growth-medium composition. Likewise, a large increase of >100-fold in nrdHIEF mRNA was observed in bacteria lacking Trx1 and Grx1, the two main NrdAB reductants. Moreover, nrdHIEF expression was triggered in response to oxidative stress, particularly in mutants missing hydroperoxidase I and alkyl-hydroperoxide reductase activities (69.7-fold) and in cells treated with oxidants (up to 23.4-fold over the enhanced transcript level possessed by cells grown on minimal medium). The mechanism(s) that triggers nrdHIEF expression remains unknown, but our findings exclude putative global regulators like RpoS, Fis, cAMP, OxyR, SoxR/S, or RecA. What we have learned about nrdHIEF expression indicates strong differences between its regulation and that of the nrdAB operon and of genes coding for components of both thioredoxin/glutaredoxin pathways. We propose that E. coli might optimize the responses to different stimuli by co-evolving the expression levels for its multiple reductases and electron donors.

Escherichia coli↗

PubMed2000

Transcriptional regulation of glutaredoxin and thioredoxin pathways and related enzymes in response to oxidative stress.

We examined the in vivo expression of up to 16 genes encoding for components of both glutaredoxin and thioredoxin systems and for members of the OxyR and SoxRS regulons. We demonstrated that grxA (Grx1) transcription is triggered in bacteria lacking Trx1 (trxA) and GSH (gshA) in an OxyR-dependent manner. We also indicated that, unlike OxyR, SoxR is not constitutively activated in the oxidizing environment of trxA gshA mutants. We discovered that the lack of Trx1 plus GSH increases the steady-state levels of Trx reductase (trxB) and Trx2 (trxC) transcripts. This increase and the trxB and trxC up-regulation caused by the constitutive oxyR2 allele indicate that OxyR also plays a role in the regulation of the thioredoxin pathway. On the contrary, no change in the expression of genes for Trx1, Grx2, and Grx3 was observed. Transcription of nrdAB (RRase) was not induced by oxidative stress yet was induced by hydroxyurea (RRase inhibitor). Induction level was as the enhanced nrdAB basal expression of trxA grxA mutants, indicating that RRase operation without Trx1 and Grx1 must lead to disturbances sensed as those caused by hydroxyurea. We also demonstrated an inverse relation between nrdAB expression and that of genes coding for components of both glutaredoxin (grxA, gorA) and thioredoxin (trxB, trxC) systems.

Bacterial Proteins↗

PubMed2000

Oxidative mutagenesis in Escherichia coli strains lacking ROS-scavenging enzymes and/or 8-oxoguanine defenses.

Escherichia coli strains with different combinations of null mutations in the katG, katE, sodA, sodB, fpg, and mutY genes were constructed to compare their spontaneous mutation frequencies and sensitivities to various oxidants with those of bacteria solely deficient in catalase (katG katE) or cytosolic superoxide dismutase (sodA sodB) and the parental strain possessing a full complement of these enzymes. The MutY DNA glycosylase represented the major protection against the mutagenic consequences of processes associated with normal aerobic metabolism. Spontaneous mutagenesis in MutY-lacking bacteria was not influenced by the absence of (A)BC excinuclease or the presence of MucAB proteins, a result consistent with 8-oxoguanine being a principal premutational lesion. In contrast, catalase and SOD represented the major protection against the genotoxic consequences of bursts of oxidative stress caused by reactive-oxygen-generating compounds. Therefore, only bacteria simultaneously defective in both katG and katE or sodA and sodB genes were hypersensitive with respect to mutability by peroxide and superoxide, respectively. These data suggest that oxidative lesions other than 8-oxoguanine contribute to mutagenesis by hydrogen peroxide and redox-cycling chemicals.

DNA Repair↗

PubMed2000

Hydrogen peroxide activates the SoxRS regulon in vivo.

By multiplex reverse transcription-PCR, we demonstrate that the SoxRS response, which protects cells against superoxide toxicity, is triggered also by hydrogen peroxide. SoxR-dependent inductions of 7. 3-, 7.6-, 4.6-, 2.2-, and 2.6-fold were quantified for soxS, micF, sodA, inaA, and fpr transcripts, respectively. This finding suggests an extensive and tight connectivity between different regulatory pathways in the Escherichia coli response to oxidative stress.

Bacterial Proteins↗

PubMed1999

Human O(6)-alkylguanine-DNA alkyltransferase: protection against alkylating agents and sensitization to dibromoalkanes.

O(6)-alkylguanine-DNA alkyltransferase (AGT) is a suicide protein that corrects DNA damage by alkylating agents and may also serve to activate environmental carcinogens. We expressed human wild-type and two active mutant AGTs in bacteria that lack endogenous AGT and are also defective in nucleotide excision repair, to examine the ability of the AGTs to protect Escherichia coli from DNA damage by different types of alkylating agents and, oppositely, to sensitize cells to the genotoxic effects of dibromoalkanes (DBAs). Control bacteria carrying the cloning vector alone were extremely sensitive to mutagenesis by low, noncytotoxic doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Expression of human wild-type AGT prevented most of this enlarged susceptibility to MNNG mutagenesis. Oppositely, cell killing required much higher MNNG concentrations and prevention by wild-type AGT was much less effective. Mutants V139F and V139F/P140R/L142M protected bacteria against MNNG-induced cytotoxicity more effectively than the wild-type AGT, but protection against the less stringent mutagenesis assay was variable. Subtle differences between wild-type AGT and the two mutant variants were further revealed by assaying protection against mutagenesis by more complex alkylating agents, such as N-ethyl-N-nitrosourea and 1-(2-chloro- ethyl)-3-cyclohexyl-1-nitrosourea. Unlike wild-type and V139F, the triple mutant variant, V139F/P140R/L142M was unaffected by the AGT inhibitor, O(6)-benzylguanine. Wild-type AGT and V139F potentiated the genotoxic effects of DBAs; however, the triple mutant virtually failed to sensitize the bacteria to these agents. These experiments provide evidence that in addition to the active site cysteine at position 145, the proline at position 140 might be important in defining the capacity by which AGTs modulate genotoxicity by environmentally relevant DBAs. The ability of AGTs to activate dibromoalkanes suggests that this DNA repair enzyme could be altered, and if expressed in tumors might be lethal by enhancing the activation of specific chemotherapeutic prodrugs.

Alkylating Agents↗

PubMed1999

Hydrogen peroxide and coffee induce G:C-->T:A transversions in the lacI gene of catalase-defective Escherichia coli.

The mutagenicity of hydrogen peroxide (H2O2) was compared with that of coffee, a complex mixture which generates H2O2. An Escherichia coli strain defective in catalase activity (katG katE double mutant) and carrying a single copy mucAB (pRW144) plasmid was constructed to enhance the mutagenic response to oxidants. The ability of the mucAB genes to influence the type, frequency and distribution of H2O2-induced mutations was also investigated in isogenic bacteria lacking pRW144. Induced mutational spectra were characterized and compared with that of spontaneous mutagenesis. A total of 444 independent forward mutations affecting the first 210 bp of the lacI gene were identified by DNA sequence analysis. The spontaneous mutation spectrum showed no bias (P = 0.52) for substitutions at G:C base pairs. In contrast, in the H2O2-induced spectrum substitutions occurred preferentially at G:C base pairs (P < 0.0001) with a preponderance of G:C-->T:A transversions (43.4% of H2O2-induced mutants versus 17.3% of spontaneous mutants). These data support the view that 7,8-dihydro-8-oxoguanine is the main premutagenic lesion induced by H2O2 and that catalase-defective bacteria have elevated levels of 8-oxoguanine in chromosome DNA after H2O2 exposure. Coffee produced a similar distribution of mutational events as H2O2 (P > 0.05), suggesting that this compound may be the main cause of the coffee-induced mutagenesis. The presence of plasmid pRW144 did not affect the frequency of H2O2-induced G:C-->T:A transversions, but caused an increase in A:T-->T:A transversions and a decrease in -1 base frameshifts. Although the frequencies of G:C-->T:A transversions were similar in all three induced spectra (H2O2 and coffee +/- pRW144), differences were observed in location of mutations throughout the target gene.

Acatalasia↗

PubMed1999

In vivo transcription of the Escherichia coli oxyR regulon as a function of growth phase and in response to oxidative stress.

Simultaneous expression of seven genes in Escherichia coli was measured by a reverse transcription-multiplex PCR fluorescence procedure. Genes studied were (i) oxyR (transcriptional regulator); (ii) katG, dps, gorA, and ahpCF (controlled by OxyR); (iii) sodA (controlled by SoxRS); and (iv) trxA (not related to OxyR or SoxRS). Except for trxA, transcription of all genes was activated during the course of growth of wild-type bacteria, though notable variations were observed with respect to both the time and extent of activation. Whereas oxyR, katG, dps, and gorA were activated during exponential growth, ahpCF and sodA were stimulated in stationary phase. Maximal induction ranged from 4.6- to 86.5-fold, for gorA and dps, respectively. Treatment with H2O2 stimulated expression of the genes (katG, dps, ahpCF, and gorA) previously identified as members of the OxyR regulon, except for oxyR itself. Induction by H2O2 was a remarkably rapid and reversible process that took place in an OxyR-dependent and sigmaS-independent manner. NaCl induced expression of the genes controlled by OxyR, including the oxyR locus. This transcriptional up-regulation was preserved in a strain with the DeltaoxyR::kan mutation, but it was abolished (ahpCF) or significantly reduced (oxyR and dps) in a strain with the rpoS::Tn10 mutation, potentially reflecting positive transcriptional regulation of the oxyR regulon by sigmaS. Expression of trxA was not increased either by H2O2 stress or by a shift to high-osmolarity conditions.

Adaptation, Biological↗

PubMed1998

In vivo transcription of nrdAB operon and of grxA and fpg genes is triggered in Escherichia coli lacking both thioredoxin and glutaredoxin 1 or thioredoxin and glutathione, respectively.

We have previously described () that Escherichia coli maintains a balanced supply of deoxyribonucleotides by a regulatory mechanism that up-regulates the levels of ribonucleotide reductase with the lack of its main hydrogen donors thioredoxin, glutaredoxin 1, and glutathione (GSH). By using a semi-quantitative reverse transcription/multiplex polymerase chain reaction fluorescent procedure that enables simultaneous analysis of up to seven mRNA species, we now demonstrate that regulation operates at the transcriptional level. Double mutant cells lacking both thioredoxin and glutaredoxin 1 had increased transcription of the nrdAB operon, as compared with the corresponding wild type parent (maximal induction of 10- and 9-fold for mRNA of nrdA and nrdB genes, respectively). Likewise, a dramatic increase of 36-fold in grxA mRNA was observed in bacteria simultaneously deficient in thioredoxin and GSH (the physiological reductant of all glutaredoxins). The increased expression of the grxA gene in trxA gshA double mutant bacteria was mimicked in trxA single mutant cells by depletion of GSH with diethylmaleate (DEM). This induction of grxA transcription was rapid since maximal increase was detected upon 10 min of DEM exposure. Like grxA expression, the basal level of fpg mRNA, encoding formamidopyrimidine-DNA glycosylase, was increased (about 4-fold) in a trxA gshA double mutant strain; this expression was also induced upon exposure to DEM (11-fold maximal induction). These results suggest that transcription of grxA might share common redox regulatory mechanism(s) with that of the fpg gene, involved in the repair of 8-oxoguanine in DNA.

Bacterial Proteins↗

PubMed1998

Mutagen content and metabolic activation of promutagens by molluscs as biomarkers of marine pollution.

Organisms combat pollutants by inducing biotransformation pathways, which can be used for biomonitoring. Several parameters--biomarkers--change in stressed organisms or populations at different organisation levels. Molecular or cellular biomarkers are early-warning indicators of pollution. Xenobiotics are first biotransformed by phase I enzymes and then conjugated with endogenous metabolites by phase II enzymes. Many organic xenobiotics are initially biotransformed by cytochrome P4501A1; in mammals, it is induced by pollutants via Ah receptor, although in marine invertebrates, its inducibility is much more equivocal. Metallothioneins are small Cys-rich proteins which bind transition metals; they detoxicate pollutant metals and are clearly induced in metal-exposed marine invertebrates. Some pollutants are genotoxins or can be converted into them. Determination of mutagens in bivalve molluscs following extraction with solvents and assay of mutagenicity with bacterial tests is a useful biomarker for marine pollution. While some pollutants are directly mutagenic, others are only mutagenic after they are activated to mutagenic derivatives by monooxygenases or conjugative enzymes. Many of these catalysts are induced by xenobiotics; thus, increased activation of known promutagens can be used as biomarker of environmental pollution. Bioactivation of promutagens requires the simultaneous action of different pathways, thus, reproducing more closely the in vivo situation than the specific assay of individual biotransforming enzymes. Study of molluscs with different pollution levels indicates that polluted animals have higher capacity to activate 2-aminoanthracene and contain more apolar mutagens than those from reference areas.

Animals↗

PubMed1998

Mutagenic activation of aromatic amines by molluscs as a biomarker of marine pollution.

Mutagenic activation of arylamines by mollusc S9 fractions was evaluated as a biomarker for marine pollution. Two bivalve species were used as bioindicators, the common mussel (Mytilus edulis) and the striped venus (Chameleo gallina). A strain of Salmonella typhimurium overproducing O-acetyltransferase was used as indicator of mutagenicity. Mussels from an area of the North Atlantic Spanish zone that was exposed to an accidental crude oil spill were compared to bivalves from a reference area. C. gallina samples were from low polluted and highly polluted areas of the South Atlantic Spanish littoral. The promutagen 2-aminoanthracene (2-AA) was activated to mutagenic derivative(s) by S9 fractions from both C. gallina and M. edulis. Animals from contaminated sites showed higher arylamine activation capabilities than reference animals. This was further correlated with the mutagenic activities of corresponding cyclopentone-dichloromethane animal extracts. 2-AA activation by mollusc S9 was potentiated by alpha-naphthoflavone (ANF), known to inhibit PAH-inducible CYP1A cytochromes from vertebrates, but inhibited by methimazole (MZ), a substrate of the flavin monooxygenase (FMO) system. 2-AA-activating enzymes were mainly cytosolic; this localization clearly suggests that such activity could be attributed to soluble enzymes, different from the CYP1A or FMO systems. In conclusion, mutagenic activation of arylamines by mollusc S9, using as indicator a strain of Salmonella typhimurium that overproduces O-acetyltransferase, could be a reliable biomarker for marine pollution.

2-Acetylaminofluorene↗

PubMed1998

What is the relationship between spermatozoa per milliliter at adulthood and the tubular fertility index at surgical age for patients with cryptorchidism?

PURPOSE: The aim of this study was to find out the relationship in the patient with cryptorchidism between the number of spermatogonia at surgical age and spermiogram data at adulthood. METHODS: Between 1971 and 1996 the authors surgically treated 1,550 children for 2,249 undescended testis. In 21 unilateral and 41 bilateral cryptorchidism patients, both testis were biopsied at surgical procedure during childhood. We currently have spermiogram data for a patient at adulthood. RESULTS: By using the best Tubular Fertility Index (TFI) figures of both testis, the Sperman correlation index between the two parameters is r = 0.22, statistically nonsignificant. The TFI test sensitivity is 0.68 and the specificity is 0.60. The likehood ratio for a positive test result is 0.84 and the likehood ratio for a negative test result is 0.37. CONCLUSION: Although the authors have only one spermiogram, and there are few cases, they suspect the TFI is not a good index to predict the potential fertility in cryptorchidism patients.

Adult↗

PubMed1998

DNA sequence analysis of spontaneous lacI-d mutations in O6-alkylguanine-DNA alkyltransferase-proficient and -deficient Escherichia coli.

Spontaneous mutagenesis in O6-alkylguanine-DNA alkyltransferase-proficient and -deficient (ada ogt mutants) Escherichia coli was studied in two ways: in bacteria growing in nonselective liquid medium and in bacteria resting on selective agar plates. ATase mutants showed similar spontaneous mutation rates as ATase proficient bacteria during growth phase; an excess of mutants arising in nondividing cells. The resting-associated mutagenesis in ada + ogt + uvr- bacteria was biphasic; the high sensitive range being triggered beyond the first 6 days after plating. Contrarily, spontaneous Lacc mutants from ada- ogt- uvr- cells steadily increased over the 8 day period of plate incubation. These results suggested that, in the absence of nucleotide excision repair, the repair by both the Ada and the Ogt ATases is not saturated until the cells have been resting for 6 days. The spontaneous LacI-d mutation spectrum of ada + ogt + uvr- bacteria growing in non-selective liquid medium served as a baseline to determine the mutation events increased in the ATase-deficient derivative upon prolonged incubation on selective plates. The percentage of G:C-->A:T transitions, presumably driven by unrepaired O6-alkylguanine lesions, was increased at the expense of other mutation types. G:C-->A:T transitions accumulated with a pronounced 5'PuG bias, suggesting that the endogenous metabolite(s) responsible for this mutation class is an SN1 type alkylating compound(s). Accordingly, the site distribution of G:C-->A:T transitions in nondividing ATase defective bacteria showed similarities with the spectra induced by alkylnitrosoureas, particularly with those generating bulky alkylated DNA adducts.

Bacterial Proteins↗

PubMed1998

Role of DNA repair by (A)BC excinuclease and Ogt alkyltransferase in the final distribution of LacI-d mutations induced by N-butyl-N-nitrosourea in Escherichia coli.

In the absence of nucleotide excision repair, the additional deficiency of the DNA alkyltransferase (ATase) encoded by the constitutive ogt gene of Escherichia coli caused a marked increase in mutation induction by N-butyl-N-nitrosourea (BNU). Irrespective of the presence or absence of the Ogt ATase, little mutagenic response was detected in Uvr+ bacteria in the concentration range 0-8 mM BNU, indicating that most premutagenic DNA lesions induced at these concentrations are efficiently recognized and repaired by the nucleotide excision repair system. Increased susceptibility to mutagenesis by BNU was detected in Uvr- Ogt+ bacteria, but the Uvr- Ogt- double mutant exhibited much higher sensitivity. These data suggest that the Ogt ATase can replace to a great extent the repair capacity of the (A)BC excinuclease. Forward mutations induced by 6 mM BNU within the initial part of the lacI gene of E.coli were recovered from Uvr+ Ogt-, Uvr- Ogt+ and Uvr- Ogt- bacteria. A total of 454 independent mutations were characterized by DNA sequence analysis. The BNU-induced spectra were dominated by G:C-->A:T transitions, consistent with the major role of the O6-alkylguanine miscoding lesion in mutagenesis by alkylating agents. Specific sites for G:C-->A:T transitions were recovered more or less frequently in one genetic background versus the others, giving statistically significant differences among the spectra (P < 10(-6)). We examined the influence of DNA repair by (A)BC excinuclease and Ogt ATase on the 5'-flanking base associated with the BNU-induced G:C-->A:T transitions; preferences different from those previously reported for other alkylnitrosoureas were detected. We discuss how these differences might be caused by BNU producing branched chain derivatives, in addition to the expected linear chain adducts, and by possible preferences with respect to both the initial distribution of O6-butylguanine lesions and their repairability.

Bacterial Proteins↗

PubMed1998

Formation of 8-oxoguanine in cellular DNA of Escherichia coli strains defective in different antioxidant defences.

This paper examines the relationship in Escherichia coli between the in vivo content of 8-oxoguanine (8-oxoG) in chromosomal DNA and deficiencies of various key antioxidant defences. The structural genes for catalases (katG and katE), cytosolic superoxide dismutases (sodA and sodB) or formamidopyrimidine-DNA glycosylase (fpg) were inactivated to obtain bacterial strains lacking the scavenger enzymes for H2O2 or O2.- or the DNA repair protein for 8-oxoG. Wild-type bacteria showed 5-fold increased sensitivity to both lethality and mutagenesis by H2O2 in K medium (1% casamino acids and 1% glucose), as compared with nutrient broth. This higher sensitivity was associated with increased chromosomal oxidative damage, estimated as the 8-oxodG content, and with a marked decrease in both catalase and SOD activities. Bacteria lacking both cytosolic SODs (sodA sodB mutant) displayed increased 8-oxodG content in chromosomal DNA (2.8-fold that of the wild-type) when grown under standard aerated conditions. Comparatively, no significant difference in 8-oxodG content was observed in cells grown without aeration. Bacteria totally devoid of catalase activity (katG katE mutant) showed wild-type contents of 8-oxodG in chromosomal DNA when grown under aerated conditions. Nevertheless, the protective role of catalase in preventing formation of 8-oxodG in chromosomal DNA became evident under oxidative stress conditions: growth under hyperoxygenation and, particularly, following H2O2 exposure. Catalase deficiency resulted in a dramatic decrease in viability after H2O2 exposure. A deficiency of Fpg protein also sensitized E.coli to H2O2 lethality, though to lesser extent than a deficiency of catalase activity. However, the scavenger enzyme and the DNA repair protein protected equally against 8-oxoG formed in vivo upon H2O2 treatment.

Alkaline Phosphatase↗

PubMed1998

Influence of DNA repair by (A)BC excinuclease and Ogt alkyltransferase on the distribution of mutations induced by n-propyl-N-nitrosourea in Escherichia coli.

In the absence of nucleotide excision repair, the additional deficiency of the DNA alkyltransferase (ATase) encoded by the constitutive ogt gene of Escherichia coli caused a marked increment in mutation induction by N-propyl-N-nitrosourea (PNU). Irrespective of the presence or the absence of the Ogt ATase, little mutagenic response was detected in Uvr+ bacteria in the concentration range 0-8 mM PNU, indicating that most premutagenic DNA lesions induced at these concentrations are efficiently recognized and repaired by the nucleotide excision repair system. Some increased susceptibility to mutagenesis by PNU was detected in Uvr- Ogt+ bacteria, but the Uvr- Ogt- double mutant exhibited much higher sensitivity. These data suggest that the Ogt ATase can replace to a great extent the repair capacity of the (A)BC excinuclease. Forward mutations induced by 6 mM PNU within the initial part of the lacl gene were recovered from Uvr+ Ogt-, Uvr- Ogt+, and Uvr- Ogt- bacteria. A total of 439 independent mutations were characterized by DNA sequence analysis. The PNU-induced spectra were dominated by G:C-->A:T transitions, consistent with the major role of the O6-alkylguanine miscoding lesion in mutagenesis by alkylating agents. Specific sites for G:C-->A:T transitions were recovered more or less frequently in one genetic background versus the others, giving statistically significant differences among the spectra (P < 10(-6)). We examined the influence of DNA repair by (A)BC excinuclease and Ogt ATase on the 5'-flanking base and DNA-strand associated with the PNU-induced G:C-->A:T transitions. Preferences different from those previously reported for the ethylating (ENU) and methylating (MNU) analogs were detected. We indicate that these differences might be caused by the PNU possibility of giving iso-propyl adducts, in addition to the expected n-propyl adducts, and by possible preferences in the initial distribution of these lesions as well as in their repair by the (A)BC excinuclease and the Ogt ATase of E. coli.

Bacterial Proteins↗